The HER-2/neu (erbB-2) gene encodes a receptor-like tyrosine kinase (RTK) which is a member of the epidermal growth factor receptor family (Coussens et al., Science 230:1132-1139, 1985). Overexpression of HER-2/neu has been observed in tumors arising at many sites including non-small cell lung (Kern et al., Cancer Res. 50:5184-5191, 1990), colon (Cohen et al., Oncogene, 4:81-88, 1989), prostate (Arai et al., Prostate 30:195-201, 1997), ovarian, and breast (Slamon et al., Science 244: 707-712, 1989). In human breast cancer, where HER-2/neu involvement has been studied, overexpression occurs in 15-30% of the cases (Singleton and Strickler, Pathol.Annual 27 Pt 1:165-198, 1992) and predicts for significantly lower survival rate and shorter time to relapse in patients with lymph node positive disease (Slamon et al., Science 244: 707-712, 1989; Singleton and Strickler, Pathol.Annual 27 Pt 1:165-198, 1992; Slamon et al., Science 235:177-182, 1987; and Slamon et al., Science 235:177-182, 1987). The significance of HER-2/neu in node negative patients is controversial and so far its clinical utility as a prognostic indicator is limited (Slamon et al., Science 235:177-182, 1987; and Hynes et al., Biochem. Biophys. Acta 1198:165-184, 1994). Various approaches are being taken toward HER-2/neu targeted therapeutics many of which are based on antibodies specific to the extracellular domain (ECD) of the transmembrane protein, which either down regulate receptor function or target recombinant toxins with the goal of specifically killing HER-2/neu expressing tumor cells (Hynes et al., Biochem. Biophys. Acta 1198:165-184, 1994; Press et al., Progress in Clinical & Biological Research 354:209-221, 1990; and Dougall et al., Oncogene 9: 2109-2123, 1994).
In addition to the full length transmembrane product, p1 85, of the HER-2/neu gene, a truncated product corresponding to the extracellular domain (ECD) is released from breast carcinoma cells in culture by regulated proteolysis (Lin and Clinton, Oncogene 6:639-643, 1991; Zabrecky et al., J. Biol. Chem. 266: 1716-1720, 1991; and Pupa et al., Oncogene, 8:2917-2923, 1993), and is also produced from an alternative transcript (Scott et al., Mol. Cell. Biol. 13:2247-2257 1993). HER-2/neu ECD is elevated in the serum of patients with breast (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992), ovarian (Maden et al., Anticancer Res. 17:757-760, 1997), and prostate cancer (Myers et al., Int. J. Cancer 69 398-402, 1996). Several studies of breast cancers estimate that 6% or less of early stage breast cancer, about 25% of patients with metastatic and locally advanced disease, and greater than 50% of patients with recurrent metastatic disease have elevated serum ECD (Brandt-Rauf et al., Mutation Res. 333:203-208, 1995). Elevated ECD in serum is associated with overexpression of HER-2/neu in tumor tissue and also has been correlated to tumor load (Molina et al., Br. J. of Cancer 4:1126-1131, 1996; and Brodowicz et al., Oncology 54:475-481, 1997). Serum ECD is a marker of metastatic disease and may predict recurrence (Molina et al., Br. J. of Cancer 4:1126-1131, 1996), shortened survival (Brodowicz et al., Oncology 54:475-481, 1997; Kandl et al., Br. J. Cancer 70:739-742, 1994; Fehm et al., Oncology 55:33-38, 1998 and Mansour et al., Anticancer Res. 17:3101-3105, 1997), and response to antiestrogen therapy in advanced stage patients (Leitzel et al., J. Clin. Oncol. 13:1129-1135, 1995; and Yamauchi et al., J. Clin. Oncol. 15:2518-2525, 1997). Serum ECD has also been reported to neutralize the activity of anti HER-2/neu antibodies targeted to the ECD (Baselga et al., J. Clin. Oncol. 14:737-744, 1996; and Brodowicz et al., Int. J. Cancer. 73:875-879, 1997) possibly allowing escape of HER-2-rich tumors from immunological control.
Cellular fragments created by ectodomain shedding have been described for the colony stimulating factor receptor (CSF-1R) (Downing et al., Mol. Cell.Biol. 9:2890-2896, 1989), the TrkA neurotrophin receptor (Cabrera et al., J. Cell. Biol. 132 427-436, 1996), Axl receptor (O'Bryan et al., J. Biol. Chem., 270:551-557, 1995), and HER-4 (Vecchi et al., J. Biol. Chem. 271:18989-18995, 1996). However, a truncated cellular product of HER-2/neu shedding has not been identified. The truncated CSF-1R was found to have in vitro kinase activity (Downing et al., Mol. Cell. Biol. 9:2890-2896, 1989), and the cytoplasmic HER-4, induced by phorbol ester tumor promoters, had little or no kinase activity (Vecchi et al., J. Biol. Chem. 271:18989-18995, 1996) while a truncated HER-4 found in cells treated with a proteosome inhibitors was an active kinase (Vecchi et al., J. Cell Biol. 139:995-1003, 1997). Therefore, there is a need in the art to identify a truncated HER-2/neu polypeptide and determine if it has enzymatic activity in general or kinase activity in particular. Moreover, such a truncated polypeptide is likely to be a better marker for tumor diagnosis, screening and prognosis as it will be easier to assay for the polypeptide than to assay for shed ECD, which is present in a much more dilute form.
The ECD of full-length transmembrane receptors often exerts a negative regulatory constraint on their signaling activity. Engineered deletion of a region of the HER-2 ECD was found to enhance its oncogenic potency (DiFiore et al., Science 237:178-182, 1987; Hudziak et al., Proc. Natl. Acad. Sci. USA 84: 7159-7163, 1987; Segatto et al., Mol. Cell. Biol. 8:5570-5574, 1988; and Bargmann and Weinberg, EMBO J. 7:2043-2052, 1988). This has also been illustrated by engineered removal of the ECD from the epidermal growth factor (EGF) receptor and by the oncogenic potency of viral encoded v-erbB, v-kit, and v-ros, that are missing regions of the ECD found in their normal cellular counterparts (Rodrigues and Park, Curr. Opin. Genet. Dev. 4:15-24, 1994). Naturally occurring mutant EGF receptors with N-terminal truncations have been identified in several human carcinomas (Moscatello et al., Cancer Res., 55:5536-5539, 1995) and have constitutive signaling activity and enhanced oncogenic transforming activity in cell culture and animal models (Moscatello et al., Oncogene 13:85-96, 1996; and Huang, et al. J. Biol. Chem. 272:2927-2935, 1997).
Therefore, there is a need in the art to better study the HER-2/neu receptor and to determine if there are better regions of this protein available for using as a more sensitive diagnostic and prognostic indicator for breast cancer. Moreover, there is no procedure available to monitor for staging and prognosis of various adenocarcinomas, such as breast cancers, other than physically investigating adjacent tissue, such as regional lymph nodes and then sectioning the tissue by difficult histological techniques. Therefore, there is a need in the art to provide improved means for determining adenocarcinoma staging and further determining prognostic factors to guide appropriate treatment strategies. The present invention was made to address the foregoing needs in the art.